The primary goal in organ transplantation is to establish a state of immunologic tolerance to donor antigens. Transplant recipients are currently prescribed toxic immunosuppressive medications that provide nonspecific suppression and increase their risk for infections and neoplasms. An experimental approach that may provide clinically relevant insights into tolerance induction employs exposure of transplant recipients to donor antigens, prior to transplantation. In several animal models and human clinical trials, pretransplant exposure to donor antigens has been found to downregulate recipient immunologic responses to the donor antigens. The cellular and molecular mechanisms by which donor antigens inhibit recipient T cells is not known. Defining these mechanisms may provide a way to develop targeted tolerogenic therapies for clinical transplantation. Our laboratory has developed a murine transplantation model to define the molecular events that regulate the induction of T cell unresponsiveness by donor antigens. Exposure of recipient mice to donor antigens downregulates T cell responses in vitro and prolongs cardiac transplant survival. We are now defining the molecular mechanisms that account for the state of T cell unresponsiveness. The hypothesis of our proposal is that pretransplant exposure to donor antigens induces T cell unresponsiveness by actively downregulating intracellular signaling mechanisms. We will extensively analyze the cellular and molecular mechanisms that are responsible for T cell unresponsiveness in three aims. Aim 1 will define cellular mechanisms that may contribute to unresponsiveness, and determine whether active suppression is a predominate mechanism. Aim 2 will determine whether infusion of donor antigens alters cell membrane activation events in unresponsive T cells. Aim 3 will examine critical downstream effector signaling pathways and determine whether specific intracellular signaling defects are induced by the infusion of donor cells. We believe that intracellular signaling alterations that lead to alloantigen-induced tolerance may provide novel targets for a new generation of tolerizing immunosuppressive agents.